National Center for Zoonotic, Vector-Borne and Enteric Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F-36, Atlanta, GA 30341, USA.
Parasitol Res. 2010 Jan;106(2):499-504. doi: 10.1007/s00436-009-1692-6. Epub 2009 Dec 3.
Baylisascaris procyonis is a helminth parasite commonly found in North American raccoons (Procyon lotor) that is a cause of clinical neural, ocular, and visceral larva migrans in humans when infective eggs are ingested. Rapid detection of B. procyonis eggs in contaminated soil and water would assist public health analysts in evaluating risks associated with public exposure to areas of known raccoon activity. In this study, a molecular beacon probe-based real-time polymerase chain reaction (PCR) assay was developed to enable rapid and specific detection of eggs of Baylisascaris spp. The molecular beacon assay targeted the cytochrome oxidase subunit 2 (cox-2) gene of B. procyonis. To determine method sensitivity, experiments testing various egg levels (250, 25, and five eggs) were performed by seeding into 0.5-g soil samples or 0.5-mL water samples. Different soil sample types were extracted using a commercial nucleic acid extraction kit. Specificity testing using previously characterized helminth tissue specimens indicated that the assay was specific to Baylisascaris spp. Little real-time PCR inhibition was observed for most of the soil and water samples. A seed level of 250 eggs was detected for all soil types, and two seed levels (25 and five eggs) were detected for surface water samples. These results demonstrate that the reported real-time PCR assay was effective for the sensitive detection of B. procyonis in a wide range of soil types, and should be a useful tool for investigations of soil or water potentially contaminated with eggs of this parasite.
贝氏蛔虫是一种常见于北美的浣熊（Procyon lotor）体内的寄生虫，当感染性虫卵被摄入人体时，会导致人类出现临床神经、眼部和内脏幼虫移行症。快速检测受污染土壤和水中的贝氏蛔虫卵将有助于公共卫生分析师评估与已知浣熊活动区域公众接触相关的风险。在这项研究中，开发了一种基于分子信标探针的实时聚合酶链反应（PCR）检测方法，以实现对贝氏蛔虫属虫卵的快速和特异性检测。分子信标检测方法针对的是贝氏蛔虫的细胞色素氧化酶亚基 2（cox-2）基因。为了确定方法的灵敏度，通过在 0.5 克土壤样品或 0.5 毫升水样品中播种，进行了各种卵水平（250、25 和 5 个卵）的实验。使用商业核酸提取试剂盒提取不同的土壤样本类型。使用先前表征的寄生虫组织标本进行的特异性测试表明，该检测方法特异性针对贝氏蛔虫属。大多数土壤和水样中几乎没有实时 PCR 抑制现象。所有土壤类型均检测到 250 个卵的播种水平，而地表水样品则检测到两个播种水平（25 和 5 个卵）。这些结果表明，所报道的实时 PCR 检测方法可有效用于检测各种土壤类型中的贝氏蛔虫，并且应该是调查可能受这种寄生虫卵污染的土壤或水的有用工具。
